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Pin1介导高氧诱导人肺泡上皮细胞凋亡中SIRT1核-浆穿梭改变.docx

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Pin1介导高氧诱导人肺泡上皮细胞凋亡中SIRT1核-浆穿梭改变 Introduction Oxygenisessentialforhumanlife,buthighlevelsofoxygenaretoxicandcaninducecelldeathinmanycelltypes,includinglungepithelialcells.Highoxygenconcentration-inducedlunginjuryisamajorcauseofpulmonarymorbidityandmortality.Theprecisemechanismsunderlyinghighoxygen-inducedcelldeathandlunginjuryarenotunderstoodfully.However,ithasbeenreportedthathighoxygenconcentrationinducesapoptosisoflungepithelialcells,whichplaysacriticalroleinacutelunginjury.SIRT1isaNAD-dependentdeacetylasethathasbeenreportedtoprotectcellsfromapoptosisinmanyorgans,includingthelung.Inthisstudy,weaimedtoinvestigatetheroleofSIRT1inhighoxygen-inducedlungepithelialcellapoptosis. MaterialsandMethods ThehumanlungadenocarcinomacelllineA549wasusedinthisstudy.ThecellswereculturedinDulbecco'smodifiedEagle'smediumcontaining10%fetalbovineserum,penicillin(100U/ml),andstreptomycin(100μg/ml)at37°Cand5%CO2.Thecellswereexposedtohighoxygenconcentration(95%O2)for24husingahypoxiachamber.SIRT1expressionwassilencedbytransfectingthecellswithsmallinterferingRNA(siRNA)targetingSIRT1.Thecontrolcellsweretransfectedwithnon-targetingsiRNA. CellviabilitywasassessedusingtheCellCountingKit-8(CCK-8)assay.ApoptoticcellsweredetectedusingtheAnnexinV-FITC/PIapoptosisdetectionkit.WesternblottingwasperformedtoassesstheexpressionofSIRT1,cleavedcaspase-3,andp53.ImmunofluorescencestainingwasdonetoobservethesubcellularlocalizationofSIRT1. Results HighoxygenconcentrationsignificantlydecreasedcellviabilityandincreasedthenumberofapoptoticcellsinA549cells.TheexpressionofSIRT1wassignificantlydecreasedinresponsetohighoxygenconcentration.KnockdownofSIRT1expressionexacerbatedhighoxygen-inducedapoptosis.Theexpressionofcleavedcaspase-3andp53wasincreasedinresponsetohighoxygenconcentration,andSIRT1knockdownfurtherupregulatedtheexpressionofcleavedcaspase-3andp53.ImmunofluorescencestainingshowedthatSIRT1translocatedfromthenucleustothecytoplasminresponsetohighoxygenconcentration. Discussion ThepresentstudydemonstratedthatS